Milk is a whitish liquid that is produced from the mammary glands of mature female mammals after they have given birth. Mammals are warm-blooded vertebrates of the Class Mammalia, including humans, the mammals, for the purposes of the present invention, being more preferably hoofed, even-toed mammals of the Suborder Ruminantia, such as cattle, sheep, goats, deer and giraffes. Milk from cattle and goats are the preferred sources of milk apoproteins of the present invention, merely because milk from these sources is readily available on a commercial scale.
Milk serum is a term commonly used in the dairy industry to describe the clear liquid matrix within which casein micelles and butterfat globules are suspended. Milk serum from ruminants contains the milk sugar lactose; a variety of proteins including milk antibodies, lactoferrin and enzymes; and a variety of lipoproteins including beta-lactoglobulin. Milk serum is a preferred source of milk apoproteins.
Cow's milk is processed in the dairy industry to obtain either butter or cheese. Mechanical agitation is used to break the milk-fat globules to obtain butter, and casein is precipitated to obtain curd from which cheese is manufactured. The liquid residue remaining after these processes is commonly referred to as milk whey. Milk whey is essentially the milk serum, with an increased lipoprotein content arising mainly from the fat globule membrane. Milk whey is a preferred source of milk apoproteins. The term “milk serum apoprotein” as used herein is intended to embrace milk apoproteins derived from milk serum or milk whey.
There are a variety of different lipoproteins and glycoproteins in milk serum, all of which are characterised by a protein back-bone, to which lipids and/or carbohydrates are conjugated. Enzymatic hydrolysis may be used to remove the lipids and/or carbohydrates from this protein back-bone, to prepare the corresponding apoprotein. Although milk serum apoproteins have been isolated, there are no known medical uses for such milk serum apoproteins.
Lipids, or fats, include triesters of fatty acids, which may be the same or different, and glycerol, also described as tri-acylglycerols or triglycerides. Further hydrolysis may be used to break these ester bonds, thus liberating free fatty acid(s) from the tri-acylglycerols. The use of calf pregastric lipase to liberate free fatty acids from milk lipids is reported by Cynthia Q Sun et al (Chemico-Biological Interactions 140 (2002), pp185-198). This author reports the growth inhibitory properties of various free fatty acids against Enterococci, which are gram-positive, and coliform bacteria, which are gram-negative but is silent on the role of milk serum apoproteins.
Free fatty acids are known to exhibit potent antimicrobial and antiviral activity. In particular, linoleic, linolenic, caprylic and caproic acids were reported by Schuster et al (Pharmacology and Therapeutics in Dentistry 5: pp25-33; 1980) to inhibit the dental caries organism, Streptococcus mutans, and to effect a general reduction in dental plaque. According to the author, bacteria classified as grain negative are most sensitive while gram positives are least affected. Additionally, Halldor Thormar et al (Antimicrobial Agents and Chemotherapy; January 1987, pp 27-31) review the antiviral properties of free fatty acids and their monoesters, demonstrating the efficacy of polyunsaturated long-chain fatty acids and medium-chain saturated fatty acids (and their monoglyceride esters) against enveloped viruses and their relative inactivity against nonenveloped viruses, the viricidal effect being possibly by destabilising the viral envelope itself. More recently, the bactericidal activity of free fatty acids was reviewed by R. Corinne Sprong et al (Antimicrobial Agents and Chemotherapy, April 2001, pp 1298-1301)—C10:0 and C12:0 fatty acids were found to be powerful bactericidal agents. The fungicidal properties of C10:0 and C12:0 free fatty acids and their monoglycerides was described by Gudmundur Bergsson et al (Antimicrobial Agents and Chemotherapy, November 2001, pp 3209-3212).
Many potentially pathogenic bacteria are common commensals of the skin, hair and mucus membranes—they colonise these areas by adhering to the surface epithelial cell layer but are normally kept in check by the host's secretory immune system in mucus and sweat. Disease caused by these endogenous species usually arises as a result of some debilitation in the host's secretory immune capability, which allows these endogenous pathogens to proliferate.
Adhesion of pathogenic bacteria to host tissue is generally accepted as being the first stage in pathogenesis, so that the ability to block adhesion should be useful in preventing infection. The mechanism of such adhesion is varied and many organisms employ a multiplicity of both specific and non-specific factors. For example, Staphylococci secrete an extracellular teichoic acid, which binds specifically to fibronectin; Candida species employ a glycocalyx of mannoprotein; and Streptococci make use of water insoluble glucans to colonise the teeth. Because of the variety of these factors, it has long been considered impossible to devise a single inhibitor, which would be effective against the wide range of potentially pathogenic species.
The use of antibodies derived by vaccination of some donor animal has been attempted in many situations but, because of the built-in specificity, the therapeutic use of these antibodies is confined to use against the species to which they have been generated.
In all of the above published data, the use of free fatty acids to inhibit growth of a wide range of bacteria, fungi and viruses is disclosed but there are no known published data disclosing or suggesting their efficacy when administered with one or more milk apoproteins in the inhibition of adhesion and/or growth of potential pathogens in human and animal healthcare.
The common practice in medical and veterinary care of infection is the application of an antibiotic substance designed to inhibit the infectious agent, which may be fungal, bacterial (both embraced by the term “microbial”) or viral. In long-term use, many antibiotic substances have lost their potency due to the evolution of resistance by the infectious agent. The problem of antibiotic resistance is most acute in post-operative situations where the infectious agent is a common inhabitant of the skin and respiratory tract and, as such, it may have been exposed to frequent and varied antibiotics over time, allowing it to evolve resistance to these substances. Large numbers of these normally innocuous agents may be disseminated during surgical or nursing procedures and may give rise to infections when the immune tolerance of the patient has been weakened by disease or extended medical intervention; such infections are frequently described as nosocomial infections.
One such nosocomial infection is commonly referred to as MRSA (methicillin resistant Staphylococcus aureus). Staphylococcus aureus is a common inhabitant of the respiratory tract of many individuals, where it is carried asymptomatically without normally causing infection. Because of its ubiquitous nature, it is thought to have been exposed to many of the commonly used antibiotic substances, and strains now exist which are resistant to all commonly used antibiotics including methicillin. Vancomycin is ‘the drug of last resort’ in MRSA, but strains have recently emerged that are resistant to vancomycin. In addition, vancomycin resistant Enterococcus faecalis (VREF) is a common inhabitant of the gut and may be disseminated from there during surgical procedures, giving rise to other nosocomial infection.
Horizontal gene transfer is a biological term used to describe the potential transfer of genetic resistance from one species to another. The transfer of antibiotic resistance from species such as VREF to pathogenic species such as Clostridium difficile (Pseudomembranous colitis) is a potentially disastrous event and one which gives cause for great concern among the medical profession.
There is therefore a great need for new antimicrobial substances, which may be used to treat such antibiotic resistant infections and others that are refractive to conventional treatments, and for new antiviral substances to treat viral infections for which there are currently few effective therapeutic remedies.
It is an object of the present invention to retard, preferably block, adhesion of pathogenic organisms and, thus, prevent or treat microbial or viral infection of the human or animal body.
It is a further object of the present invention to combine the retarding or blocking of adhesion with an inhibition of growth, thereby achieving an even greater utility.
It is a still further object of the present invention to achieve these utilities by the use of a benign material such as, but not limited to, milk serum since this facilitates much more frequent use than is considered prudent with many aggressive chemically based medicines.